The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the ␤ sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883-5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303-P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.mutagenesis ͉ toolbelt ͉ translesion DNA synthesis ͉ Pol IV ͉ DinB E ndogenous and exogenous agents continuously damage cellular DNA. As a result, faithful duplication of an organism's genetic information requires both high fidelity DNA polymerases (Pols), as well as a multitude of DNA repair and DNA damage tolerance mechanisms (reviewed in ref.

Translesion synthesis (TLS) by Y-family DNA polymerases alleviates replication stalling at DNA damage. Ring-shaped processivity clamps play a critical but ill-defined role in mediating exchange between Y-family and replicative polymerases during TLS. By reconstituting TLS at the single-molecule level, we show that the Escherichia coli β clamp can simultaneously bind the replicative polymerase (Pol) III and the conserved Y-family Pol IV, enabling exchange of the two polymerases and rapid bypass of a Pol IV cognate lesion. Furthermore, we find that a secondary contact between Pol IV and β limits Pol IV synthesis under normal conditions but facilitates Pol III displacement from the primer terminus following Pol IV induction during the SOS DNA damage response. These results support a role for secondary polymerase clamp interactions in regulating exchange and establishing a polymerase hierarchy.single-molecule techniques | DNA replication | DNA repair | lesion bypass | DinB

SUMMARY Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the E. coli β clamp interact physically with the DNA that it topologically encircles. We utilized mutant β clamp proteins bearing G66E and G174A substitutions (β159), affecting the single strand (ss) DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (β148–152), affecting the double strand (ds) DNA binding region, in order to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the x-ray crystal structure of β148–152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both β159 and β148–152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of β148–152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired β148–152-DNA interactions. Once loaded, β148–152 was proficient for Pol III replication in vitro. In contrast, β148–152 was severely impaired for Pol II and Pol IV replication, and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, β148–152 was unable to support viability of E. coli. Nevertheless, physiological levels of β148–152 expressed from a plasmid efficiently complemented the temperature sensitive growth phenotype of a strain expressing β159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.

Translesion DNA synthesis (TLS) by specialized DNA polymerases (Pols) is a conserved mechanism for tolerating replication blocking DNA lesions. The actions of TLS Pols are managed in part by ring-shaped sliding clamp proteins. In addition to catalyzing TLS, altered expression of TLS Pols impedes cellular growth. The goal of this study was to define the relationship between the physiological function of Escherichia coli Pol IV in TLS and its ability to impede growth when overproduced. To this end, 13 novel Pol IV mutants were identified that failed to impede growth. Subsequent analysis of these mutants suggest that overproduced levels of Pol IV inhibit E. coli growth by gaining inappropriate access to the replication fork via a Pol III-Pol IV switch that is mechanistically similar to that used under physiological conditions to coordinate Pol IV-catalyzed TLS with Pol III-catalyzed replication. Detailed analysis of one mutant, Pol IV-T120P, and two previously described Pol IV mutants impaired for interaction with either the rim (Pol IVR) or the cleft (Pol IVC) of the β sliding clamp revealed novel insights into the mechanism of the Pol III-Pol IV switch. Specifically, Pol IV-T120P retained complete catalytic activity in vitro but, like Pol IVR and Pol IVC, failed to support Pol IV TLS function in vivo. Notably, the T120P mutation abrogated a biochemical interaction of Pol IV with Pol III that was required for Pol III-Pol IV switching. Taken together, these results support a model in which Pol III-Pol IV switching involves interaction of Pol IV with Pol III, as well as the β clamp rim and cleft. Moreover, they provide strong support for the view that Pol III-Pol IV switching represents a vitally important mechanism for regulating TLS in vivo by managing access of Pol IV to the DNA.

The dnaN159 allele encodes a temperature-sensitive mutant form of the ␤ sliding clamp (␤159). SOS-induced levels of DNA polymerase IV (Pol IV) confer UV sensitivity upon the dnaN159 strain, while levels of Pol IV ϳ4-fold higher than those induced by the SOS response severely impede its growth. Here, we used mutations in Pol IV that disrupted specific interactions with the ␤ clamp to test our hypothesis that these phenotypes were the result of Pol IV gaining inappropriate access to the replication fork via a Pol III*-Pol IV switch relying on both the rim and cleft of the clamp. Our results clearly demonstrate that Pol IV relied on both the clamp rim and cleft interactions for these phenotypes. In contrast to the case for Pol IV, elevated levels of the other Pols, including Pol II, which was expressed at levels ϳ8-fold higher than the normal SOS-induced levels, failed to impede growth of the dnaN159 strain. These findings suggest that the mechanism used by Pol IV to switch with Pol III* is distinct from those used by the other Pols. Results of experiments utilizing purified components to reconstitute the Pol III*-Pol II switch in vitro indicated that Pol II switched equally well with both a stalled and an actively replicating Pol III* in a manner that was independent of the rim contact required by Pol IV. These results provide compelling support for the Pol III*-Pol IV two-step switch model and demonstrate important mechanistic differences in how Pol IV and Pol II switch with Pol III*.